PROJECT ABSTRACT The OVERALL OBJECTIVES of this proposal are to unravel the mechanisms by which hepatic macrophages promote immune tolerance to cholangiocarcinoma (CCA) with the therapeutic goal of defining combination im- mune-directed therapies to treat this highly lethal cancer. A desmoplastic malignancy, CCA is characterized by a rich multi-cellular, tumor stroma, including an abundance of pro-tumor macrophages. We have established two unique CCA murine models with desmoplasia: i) an oncogene-driven model to study CCA carcinogenesis, and ii) a syngeneic, orthotopic model to study CCA progression. We are now poised to exploit these models to examine the tumor immune microenvironment (TIME) in CCA initiation and progression. Our preliminary data suggest that expression of the immune checkpoint molecule programmed cell death ligand-1 (PD-L1) on host immune cells of the TIME may be more potent than cancer cell expressed PD-L1 in facilitating tumor progres- sion. In our preliminary experiments, we have discovered that although murine CCA cells have abundant PD- L1 expression, orthotopic implantation of these cells into the liver of PD-L1 knockout mice results in a signifi- cantly reduced tumor burden, compared with wild type mice. Our preliminary data also demonstrate that he- patic macrophages, including resident Kupffer cells express PD-L1 in CCA, and CCA cell-derived extracellular vesicles (EVs) increase PD-L1 expression in hepatic macrophages. Finally, we have demonstrated that macrophage-directed therapy, with colony stimulating factor 1 receptor (CSF1R) inhibition or checkpoint inhibi- tion with PD-L1 blockade is tumor suppressive. Based on these novel observations, we have formulated the CENTRAL HYPOTHESIS of the proposal that the liver manifests a pro-tumor immune tolerogenic niche in CCA, mediated by PD-L1 positive hepatic macrophages, which can be overcome by combination immune- directed therapies reprogramming macrophages as an anti-tumor therapeutic strategy. Our following inde- pendent SPECIFIC AIMS will test three integrated hypotheses. First, we will directly test the hypothesis that CCA cell derived EVs contain oncogenic microRNAs that reprogram hepatic macrophages to a PD-L1 positive, pro-tumor phenotype. Second, we will test the hypothesis that PD-L1 positive, pro-tumor hepatic macrophages mediate intrahepatic T lymphocyte exhaustion. Third, using our murine models of CCA carcinogenesis and progression, we will test the hypothesis that the combination of macrophage-directed therapy plus checkpoint inhibition restrains both CCA carcinogenesis and tumor progression. We propose to employ current and com- plementary molecular and cell biological approaches, as well as biophysical technologies such as flow cytom- etry and mass cytometry to test this hypothesis. This proposal, which is technically and conceptually innovative, is also highly significant because it identifies new mechanisms for therapeutically targeting CCA, namely combining therapies promoting both cytotoxic T cell and macrophage anti-tumor activity.